Two gonococcal transferrin (Tf)-binding proteins, Tbp1 and Tbp2, are surface-exposed and potential vaccine candidates. The overall goal of these studies is to determine how Tbp1 and Tbp2 are organized on the cell surface and how they function together to bind and subsequently transport Tf-bound iron through the outer membrane. The specific aims are to address the following questions: 1.) What domains of Tbp1 and Tbp2 are on the surface of N. gonorrhoeae? Are any of these surface-exposed domains necessary for Tf binding? Deletion, epitope-insertion and site-specific mutations in Tbp1 and Tbp2 will be generated and assessed for their effects on Tf-binding and Tf-iron utilization. Reporter epitopes, inserted at various locations in Tbp1 and Tbp2, will be tested for their surface exposure by antibody binding to intact cells. 2.) Do Tbp1 and Tbp2 interact to form a complex and if so, what is the stoichiometry? Non-denaturing polyacrylamide gels and immunoprecipitation will be utilized to assess complex formation. If Tbp1 and Tbp2 complexes are identified, stoichiometry estimates will be made by quantitation of these proteins following denaturing gel electrophoresis of isolated complexes. 3.) Does Tbp1 form a pore, and if so, does the presence of Tbp2 affect the pore? Native and deleted versions of Tbp1 will be tested for their effect on permeability of proteoliposomes in a liposome swelling assay. Proteoliposomes will also be reconstituted in the presence and absence of Tbp2 to determine if this protein effects liposome permeability. These studies will lead to an understanding of the surface topology and functional organization of the gonococcal Tf receptor and will be useful in evaluating these antigens as potential vaccine candidates.